Holoprosencephaly (HPE) is the most common embryologic malformation of the forebrain in humans caused by incomplete cleavage of the prosencephalon. This malformation which affects the development of the prechordal plate and anterior neuroectoderm includes various degrees of midline fusion and cyclopia affecting the forebrain and face. Various genetic factors and environmental agents contribute to the etiology of HPE. In humans, mutations in the SIX3 gene encoding a homeodomain transcription factor have been associated with HPE. The genetic and cellular mechanisms of SIX3-promoted HPE are poorly understood. It remains unclear whether mutant SIX3 proteins have hypomorphic, antimorphic, or neomorphic activity. SIX3 mutations cause HPE in a dominant manner but with variable penetrance and expressivity, a finding that suggests that S/X3 interacts with other genetic loci. Functional inactivation of Six3 in mice has shown that repression of Wnt signaling in the anterior neuroectoderm is essential for vertebrate forebrain development; however, S/x3-heterozygous mice did not exhibit any obvious morphologic alteration. In this application, we propose to employ a combination of genetic, embryologic, and molecular methods to reproduce and characterize the HPE/cyclopia phenotype in mouse and zebrafish. Aim 1 entails in vivo and in vitro molecular and transcriptional characterization of the generated HPE Six3 mutant proteins. Aim 2 will generate zebrafish and mouse models of Six3-mediated HPE. We will use these models to identify tissues and'genetic pathways affected by mutant Six3. Aim 3 focuses on the identification of genes that cooperate with mutated Six3 in promoting HPE. These proposed studies will advance our understanding of the signaling pathways affected by HPE-Six3 mutations and, ultimately, will provide additional information to be used with the genetic counseling of human carriers of HPE-SIX3 mutations and decrease the frequency of these birth defects.